Proper regulation of the cellular response to hypoxia is of critical importance for the maintenance of cellular and organismal viability and function. Although much has been learned about the cellular hypoxic response in the last twenty years, several fundamental gaps in our understanding of oxygen sensing still exist. Our previous work has shown that production of mitochondrial Reactive Oxygen Species (ROS) during the hypoxic response is critical for the induction of the Hypoxia Inducible transcription Factor (HIF). It is unknown however, how mitochondria produce increased levels of ROS under conditions of low oxygen. We will test the ability of isolated mitochondria to produce ROS in response to low oxygen. Because our work has demonstrated that the QO site of mitochondrial complex III is critical for ROS production during hypoxia, we will also determine the ability of isolated mitochondrial complex III to produce ROS under conditions of low oxygen. Our preliminary data demonstrates that ROS regulate HIF through the inhibition of the Prolyl Hydroxylase Domain protein 2 (PHD2), which targets HIF for degradation. How ROS regulate PHD2 remains unknown. We will therefore examine the role of the amino-terminal (non-catalytic) domain of PHD2 In the regulation of PHD2 function during hypoxia. Previous reports suggest that this is an inhibitory regulatory domain, although the mechanism of inhibition is unexplored. Finally, we will create the first mouse model of mitochondrial oxygen sensing to demonstrate the role of mitochondrial ROS in HIF regulation in vivo. Epidermis acts as an oxygen sensor by regulating blood flow to the kidneys, which produce erythropoietin during hypoxia. We will therefore conditionally knock out the mitochondrial transcription factor TEAM in keratinocytes, thus depleting mitochondria and ROS production in these epidermal cells. We will then subject these mice to hypoxia to demonstrate the role of mitochondria as oxygen sensors in vivo. Relevance to Public Heath: While healthy individuals encounter hypoxia at high altitudes, pathological conditions of hypoxia arise during Ischemia-reperfusion injury, heart and lung disease, and cancer. Because these conditions represent major causes of mortality, further study of the mechanisms by which cells respond to hypoxia is needed. We will thus further explore the role of mitochondrial ROS in regulating the cellular hypoxic response.